Re: IHC Question

Patsy wrote:
I have been having trouble with non-specific background staining and have
gone to putting acetylated BSA in my wash buffer, which helps. My question
is: is there any reason not to also put the TWEEN in the wash buffer, it
seems that I read something a while back suggesting that they should not be
used together??????
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Dear Patsy,
Regular, non-modified, BSA predominantly prevents hydrophobic background.
It is used in a blocking step (combined with e.g. normal serum) before the
actual immuno-incubation. Tween 20 is also used to prevent hydrophobic
background by encapsulating (micelle formation) antibodies and other
immunoreagents. It will however remove the proteins used in the blocking
step and thus this step can be left out the incubationprocedure when using
Tween 20. As already written by Karen Larisonin a previous answer to your
question Tween has to be used above the critical micelle concentration (in
practice 0.1%).
BSA only has minor effect on prevention of charge dependant background
(when used in the washing buffer). Acetylated BSA (BSA-c) however does.
This effect is still present when BSA-c is used in combination with Tween
20. A combination of BSA-c and Tween 20 is therefore possible. Suggested
wash buffer composition:
PBS + 0.1% BSA-c + 0.1% Tween 20, pH 7.4.
General remark: Tween 20 is a detergent and might wash out your antigen.
Hope this info is of help.
Peter
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Peter van de Plas
AURION
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